This application is a 371 of PCT/FR98/01583, filed Jul. 20, 1998.
The invention relates to new antimicrobial peptides produced by penaeid prawns.
Peptides endowed with antimicrobial properties are produced by a wide variety of (animal or plant) species in which they participitate in nonspecific mechanisms of defence against infections. These peptides are the subject of increasing interest, in particular because they generally possess a broad activity spectrum and a low cytotoxicity for eukaryotic cells.
Comparisons of the amino acid sequences, of the secondary structures and the functional similarities have made it possible to define four main groups to which most of the antimicrobial peptides described up until now can be attached. For a review, cf. for example HOFFMANN et al., in Phylogenetic Perspectives in Immunity; The Insect Host Defense. Chapter 4, pp. 43-65 (1994):
1. A first group comprises linear peptides consisting essentially of basic and hydrophobic amino acids; the cecropins of insects and of mammals, and the magainins of the skin of batrachians, in particular are classified in this group;
2. A second group comprises peptides comprising intramolecular disulphide bridges; the defensins of insects or of mammals, the brevinins of the skin of batraciens, the thanatin of insects, which exhibits a strong sequence homology with the brevinins [FEHLBAUM et al. Proc. Natl. Acad. Sci. USA. 93, pp. 1221-1225, (1996)], the tachyplesins, produced by primitive marine arthropods, as well as various peptides obtained from the haemolymph of scorpions [EHRET-SABATIER et al., J. Biol. Chem., 271, 47, pp. 29537-29544, (1996)], in particular are classified in this group;
3. The third group comprises the peptides rich in proline, among which there may be classified the apidaecins and the abaecins of hymenopterans, drosocin and pyrrhocoricin, also produced by insects, and the bactenecins of mammals. The only antimicrobial peptide produced by a decapod crustacean which had been characterized up until now also belongs to this class: it is an antibacterial peptide rich in proline, similar to bactenecin-7, and obtained from the haemocytes of the crab Carcinus maenas [SCHNAPP et al., Eur. J. Biochem. 240, pp. 532-539 (1996)];
4. The fourth class comprises peptides or polypeptides rich in glycine, such as attacins, sarcotoxins and diptericins, all isolated from insects.
The inventors have now purified and characterized new antimicrobial peptides from the haemolymph of penaeid prawns, and have also obtained DNA sequences encoding these peptides.
The subject of the present invention is these antimicrobial peptides, called hereinafter penaeidins, which possess the following characteristics:
their molecular mass is about 5 to 7 kDa;
their pHi is greater than or equal to 9;
their N-terminal sequence comprises a region (A) of about 15 to 25 amino acids, rich in proline, (at least xe2x85x9 and preferably between xe2x85x9 and ⅓ of the amino acids of this region are prolines);
their C-terminal portion comprises a region (B) of about 20 to 30 amino acids, which contains 6 cysteine residues forming three intramolecular disulphide bridges.
According to a preferred embodiment of the present invention, region (A) comprises the following sequence (I):
Pro Xaa1 ProXaa2 ProXaa3 Pro(I, SEQ ID NO: 15 or 19)
in which Pro represents a proline, Xaa1, represents a nonpolar neutral amino acid, Xaa2 represents a basic amino acid, Xaa3 represents a proline or a peptide bond.
According to a preferred feature of this embodiment, region (A) comprises the following sequence (II):
ProXaa1 ProXaa2 ProXaa3 ProXaa1 Xaa1 Xaa2 ProXaa4 Xaa4 (II, SEQ ID NO: 17 or 21)
in which Pro, Xaa1, Xaa2 and Xaa3 are as defined above, and Xaa4 represents a nonpolar neutral amino acid or a proline.
Advantageously, region (A) comprises the following sequence (III):
ProXaa1 ProXaa2 ProXaa3 ProXaa1 Xaa1 Xaa2 ProXaa1 Pro Xaa1 Xaa1 ProXaa1 Xaa1 Pro(III, SEQ ID NO: 18 or 22)
in which Pro, Xaa1, Xaa2 and Xaa3 are as defined above.
According to another preferred embodiment of the present invention, the 6 cysteine residues of region (B) are arranged according to the following sequence (IV):
Cys S1 Cys S2 Cys Cys S3 Cys Cys (IV, SEQ ID NO: 16 or 20)
in which Cys represents a cysteine, S1 represents an amino acid or a peptide sequence of 2 or 3 amino acids, S2 represents a peptide sequence of 10 amino acids, S3 represents a peptide sequence of 5 amino acids.
The present invention also encompasses peptides comprising or consisting of fragments of at least 5 amino acids of a penaeidin as defined above, and in particular peptides comprising or consisting of region (A) and/or region (B), as well as peptides comprising or consisting of sequences (I), (II), (III) and/or (IV).
According to a preferred embodiment of a peptide in accordance with the invention, its N-terminal end is blocked by a pyroglutamic acid residue, and/or its C-terminal end is amidated.
By way of illustration of the subject of the present invention, the characteristics of 3 penaeidins isolated from the haemolymph of the prawn Penaeus vannamei, called hereinafter penaeidin 1, penaeidin 2 and penaeidin 3, are more specifically indicated below.
xe2x80x9cThe sequences of these 3 peptides (1-letter code) are represented in FIGS. 1a (penaeidin 1, SEQ ID NO: 5), 1b (penaeidin 2, SEQ ID NO: 6 with amidated C-terminus) and 1c (penaeidin 3, SEQ ID NO: 7 with amidated C-terminus and pyroglutamic acid at the N-terminus); the alignment of the 3 sequences is represented in FIG. 1d: the conserved sequences are delimited. FIG. 2 represents a cDNA sequence of penaeidin 2 and the corresponding peptide sequence (3-letter code); FIG. 3A represents a cDNA sequence of penaeidin 3 and the corresponding peptide sequence (3-letter code); FIG. 3B represents the peptide sequence (1-letter code) of other isoforms of penaeidin 3 (P3-b and P3-c, SEQ ID NOS: 23 and 24, respectively): the sequence variations are delimited.xe2x80x9d
The cDNA sequences of penaeidin 2 and of penaeidin 3 are respectively represented in the sequence listing in the annex under the numbers SEQ ID NO: 1 and SEQ ID NO:3, and the sequences of their products of translation are respectively represented in the sequence listing in the annex under the numbers SEQ ID NO: 2 and SEQ ID NO: 4.
The peptide sequences of the mature forms of penaeidins 1, 2 and 3 are respectively represented in the sequence listing in the annex under the numbers SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
These penaeidins exhibit no significant homology with the antimicrobial peptides known in the prior art, and define a new group of antimicrobial peptides.
Penaeidins 1 and 2 comprise 50 amino acids, and have a molecular mass of about 5.5 kDa; penaeidin 3 comprises 62 amino acids and its molecular mass is about 6.6 kDa.
They are cationic peptides possessing a net positive charge of 7 for penaeidins 1 and 2, and of 8 for penaeidin 3; their calculated isoelectric points vary from 9.34 for penaeidins 1 and 2, to 9.84 for penaeidin 3.
The 3 peptides have an N-terminal domain rich in proline, and a C-terminal domain comprising 6 cysteine residues forming three intramolecular disulphide bridges, and where the 4 cysteine residues closest to the C-terminal end are organized into 2 doublets separated by 5 residues.
The most central cysteines are respectively separated by 1, 2, or 3 residues in penaeidins 1, 2 and 3.
The N-terminal end of penaeidin 3 is blocked by a pyroglutamic acid residue. Blocking by similar residues has already been observed in other antimicrobial peptides.
The C-terminal end of penaeidins 2 and 3 is amidated. Such a C-terminal amidation has already been observed for certain antimicrobial peptides of other marine invertebrates (Limulus tachyplesins), as well as for the cecropins of insects and the magainins of amphibians. Such a modification reinforces the stability of the molecule, and it appears that it increases the antimicrobial activity.
Penaeidins possess a high stability and are in particular highly resistant to proteolysis. They are essentially active against Gram-positive bacteria, and also exhibit activity against Gram-negative bacteria; they possess, in addition, fungicidal properties.
Penaeidins and their fragments, as defined above, may be obtained, for example, by extraction from animals producing them, and also by peptide synthesis, or advantageously by genetic engineering, by expressing at least one nucleic acid sequence encoding a penaeidin or a fragment thereof in an appropriate host cell.
The present invention also encompasses nucleic acids comprising a segment of at least 10 bp, and preferably at least 18 bp of the gene for a penaeidin.
Nucleic acids in accordance with the invention, and in particular cDNAs for penaeidin, or portions thereof, may be obtained by screening nucleic acid libraries with the aid of oligonucleotides derived from the sequences represented in FIGS. 1, 2 or 3, or their complementary sequences.
Nucleic acids in accordance with the invention also include expression cassettes comprising at least one nucleic acid sequence encoding a penaeidin or a fragment thereof, under transcriptional control of an appropriate promoter.
xe2x80x9cAppropriate promoterxe2x80x9d is understood to mean any promoter which is functional in the host cell designated to harbour the expression cassette.
An expression cassette in accordance with the invention may also comprise, in addition, one or more nucleic acid sequence(s) which make it possible to enhance the secretion of the penaeidin or of its fragment by the host cell, for example a sequence encoding a signal peptide. The nucleic acid sequence encoding the signal peptide is placed at the 5xe2x80x2 end of the nucleic acid sequence encoding the penaeidin or its fragment.
The nucleic acid sequence encoding the signal peptide may be a sequence encoding a penaeidin signal peptide, or alternatively a sequence encoding a heterologous signal peptide. A sequence will be chosen which comprises, at the C-terminal end, a site for proteolysis capable of being recognized by a signal peptidase of the host cell designated to harbour the expression cassette.
The subject of the invention is also:
recombinant vectors characterized in that they comprise at least one nucleic acid in accordance with the invention, and in particular the vectors comprising an expression cassette as defined above;
prokaryotic or eukaryotic cells transformed with an expression cassette according to the invention. They may be cells maintained in culture, or cells forming part of a pluricellular, animal or plant, organism. The expression cassette present in the transformed cell may be either incorporated into the chromosomal DNA of the said cell, or may be carried by an extra chromosomal vector.
The subject of the invention is also a method of producing a penaeidin or a fragment thereof, characterized in that it comprises the expression of the said penaeidin or of the said fragment in at least one transformed cell in accordance with the invention.
The peptides in accordance with the invention may be expressed in cultures of transformed cells using techniques similar to those used for antimicrobial peptides of the prior art, for example in insect cells, as described by HELLERS et al. [Eur. J. Biochem. 199, pp. 435-439, (1991)] for cecropins, or in yeast, as described by REICHHART et al. [Invertebrate Reproduction and Development, 21, pp. 15-24, (1992)].
They may also be expressed in transgenic plants or animals, in order to increase the resistance of the latter to infections, as described for example by JAYNES et al., [Plant Science, 89, pp. 43-53 (1993)] in the case of peptides which are analogues of cecropin B, expressed in transgenic tobacco plants, or by NORELLI et al. [Euphytica, 77, pp. 123-128 (1994)] for transgenic young apple trees expressing the gene of attacin-E.
The peptides in accordance with the invention can be used in particular for the production of products and in particular of medicaments against infections, for example antibacterials or fungicides.
Such products find their application for the prevention and the treatment of various microbial diseases, in a very wide variety of sectors, in particular in the fields of health and agriculture, and in that of aquaculture, for limiting the development of infectious diseases in breeding farms.